Study The Effect Of Plateau Pregnancy On Different Tissues Of Newborn Piglets Based On Mitochondrial DNA Copy Number And LncRNA Sequencing

Dreadful environment in plateau cause a strong threat to animal health,survival and reproduction,which also seriously restricts the large-scale development of plateau livestock.Meanwhile,the special geographical environment,mysterious historical culture and unique natural scenery in plateau also attract large crowds into high-land,such as exploiting high-land natural resource,building national defense,hiking adventure,plateau tourism and so on.Therefore,it is particular important to study the adaptation to high-altitude.Nevertheless,previous studies mainly based on long-term adaption of resident adults and the molecular mechanisms involved in stress reaction to high-altitude pregnancy in newborns have not yet been fully elucidated.Therefore,this study use pig to explore the difference in molecular level between newborn piglets from high-altitude pregnancy and low-altitude pregnancy.In this study,we used newborn Rongchang piglets from high-altitude pregnancy(n = 3,elevation 3,658 m)and low-altitude pregnancy(n=3,elevation 711 m)as our experimental group and control group,respectively,to analysis the differentce between experimental and control group in terms of mtDNA copy number and transcriptome.The mains results are described as follows:(1)Compared to low-altitude newborn piglets,except heart tissue(p>0.05),the mtDNA copy number of high-altitude newborn piglets in six tissues(brain,liver,lung,kidney,psoas major muscle and longissimus doris muscle)is significantly decreased(p<0.05).(2)Among these seven tissues(brain,heart,liver,lung,kidney,psoas major muscle and longissimus doris muscle),mtDNA copy number is relative higher in longissimus doris muscle,brain and heart,and relative lower in lung and kidney,show significant characteristics of tissue tendentious.(3)In this study,we constructed 18 lncRNA sequencing libraries which consist of 2 different altitude,3 tissues with 3 biological repeats per treatment by altitude and tissue,on average,yielding about 40 M(million)high-quality reads(clean reads)in each library.In total,we identified 17,264 mRNA and 13,744 lncRNA.In detail,we detected 14,604,13,208 and 14,928 mRNA,also identified 12,818,11,242 and 10,943 lncRNA expressed in brain,heart and lung,respectively.(4)Through hierarchical clustering based on the expression,both mRNA and lncRNA can separated the samples by tissue and altitude.Pearson correlation analysis based on the expression indicate that correlation between biological replicates is highest,followed by the correlation between alittudes.Notably,correlation between tissues is lowest.In summary,the expression of transcripts show obvious difference among tissues.(5)Among the 13,744 IncRNA identified in our study,9,248 IncRNA were co-expressed in brain,heart and lung.Moreover,109,44 and 40 IncRNA were specifically expressed in brain,heart and lung,respectively.(6)Compared with mRNA.IncRNA show obvious characteristics with lower expression level,shorter length,less exons and lower GC content.In view of the expression level,both mRNA and IncRNA shared the same pattern that only a few types of mRNA or IncRNA are extremely abundant highly expressed and comprised the majority of total expression,on the other hand,plenty of mRNA or IncRNA are extremely poor expressed and comprised a tiny minority of total expression.(7)We carried out difference analysis to identify differentially expressed genes(DE mRNA)between pairwise high-and low-altitude samples in each tissue.In result,511 mRNA differentially expressed in brain,which consist of 315 up-regulated mRNAs and 196 down-regulate mRNAs in high-altitude pregnancy group;574 mRNA differentially expressed in heart,which consist of 253 up-regulated mRNAs and 321 down-regulate mRNAs in high-altitude pregnancy group;529 mRNA differentially expressed in lung,which consist of 283 up-regulated mRNAs and 246 down-regulate mRNAs in high-altitude pregnancy group.(8)We carried out GO annotation and KEGG analysis of DE genes between pairwise high-and low-altitude samples in each tissue.In result,the DE genes in brain mainly enriched in brain and nerve activity(axon and dendrites,etc.),metabolism and epigenetic modification;the DE genes in heart mainly enriched in immue response;the DE genes in lung mainly enriched in cellular mitosis,inflammation response and immune.(9)According to further strict filtering based on expression and coefficient of variation,we identified 273,225 and 228 high-confidence DE mRNA in brain,heart and lung,respectively.These high-confidence DE mRNA can regarded as important candidates in subsequent function validation experiment.(10)According to genomic location and strand of lncRNA.we divided the 13,744 IncRNA into 14 categories.namely long intergenic non-coding RNA.sense upstream,sense downstream,antisense upstream,antisense downstream,sense inside intron.sense outside intron,sense overlapping intron.antisense inside intron.antisense outside intron.antisense overlapping intron.antisense outside exon,antisense inside exon,antisense overlapping exon.(11)LncRNA is unevenly distributed among the chromosomes(χ2 = 1989.65,p<10-6),which show that lncRNA trend to distribute on these chromosomes with high gene density and long chromosome length.In detail,the number of IncRNA identified on chromosome is significantly positive correlated with chromosome length(r = 0.819,p = 1.77 ×10-5),gene number(r = 0.864,p = 1.82 × 10-6),pseudogene number(r = 0.706,p = 7.39 × 10-4)and protein number(r = 0.774,p ＝ 1.01 × 10-4).(12)We carried out difference analysis to identify differentially expressed IncRNA(DE lncRNA)between pairwise high-and low-altitude samples in each tissue.In result,2,331 lncRNA differentially expressed in brain,which consist of 2,220 up-regulated IncRNA and 111 down-regulate IncRNA in high-altitude pregnancy group;115 IncRNA differentially expressed in heart,which consist of 40 up-regulated mRNA and 75 down-regulate mRNA in high-altitude pregnancy group;106 IncRNA differentially expressed in lung,which consist of 39 up-regulated IncRNA and 67 down-regulate IncRNA in high-altitude pregnancy group.(13)According to the strong correlation between protein coding genes and DE lncRNA,we predicted the function of DE IncRNA in each tissue.In result,the DE lncRNA in brain were significantly related to nerve acitivity and brain development,DE lncRNA in heart were related to cardiac contraction and "neural signal transmission",DE lncRNA inlung were significantly related to cell proliferation and immune.(14)According to further strict filtering based on expression and coefficient of variation,we identified 288,15 and 9 high-confidence DE IncRNA in brain,heart and lung,respectively.These high-confidence DE IncRNA can regarded as important candidates in subsequent function validation experiment.